Plant polysaccharide fractions inducing prolactin in mammals

ABSTRACT

A polysaccharide containing units corresponding to rhamnose, arabinose, xylose, mannose, galactose and glucose, additionally providing animals and humans with an increase in the plasma prolactin content.

This is a division of application Ser. No. 07/216,806, filed Jul. 8,1988, now U.S. Pat. No. 4,948,785.

The subject of the present invention is polysaccharide extracts, inparticular from plants, as well as procedures for extracting them andtheir use as medicines and food additives.

These novel polysaccharide substances have the property of increasingthe secretion of prolactin, the principal hormone of lactation, and ofincreasing the secretion of beta-casein, the principal protein of milk,and, as a consequence, they increase the secretion of milk in women andin animals and are thus likely to find industrial applications, inparticular in the nursing of infants and in the breeding of animals.

More particularly the present invention relates to polysaccharidescontaining units corresponding to rhamnose, arabinose, xylose, mannose,galactose and glucose and providing humans and animals with an increasedlevel of plasma prolactin.

Furthermore, these polysaccharides are characterized in that theirmolecular mass lies between 10⁴ and 10⁶ daltons, and usually liesbetween 2×10⁴ and 10⁵ daltons.

In addition, these polysaccharides contain nitrogen and sulfur, usuallyfrom 1 to 8% of nitrogen and from 0.8 to 2% of sulfur.

These products are usually of plant origin but it is possible toenvisage their synthesis or semi-synthesis starting from products ofdiverse origin or even a total synthesis starting from known chemicalcompounds.

The invention also relates to procedures for the preparation of thesepolysaccharides from plants containing these polysaccharides byextraction with hot water, the polysaccharides being recovered from theaqueous extract obtained, which can itself be purified if necessary.

The purification can be performed by all of the known procedures but, inparticular, it is possible to envisage a precipitation from the aqueousextract with the aid of organic solvents which are miscible with waterand which do not dissolve the polysaccharides, in particularhydroxylated solvents such as ethanol and methanol.

It is of course possible to contemplate other methods of purificationsuch as fractionation by molecular weight.

The products according to the present invention are extracted moreespecially from plants of the genus Gossypium sp. of the family of theMalvaceae and from the species Euphorbia Hirta Linne of the family ofthe Euphorbiaceae.

The procedure of the invention consists essentially in treating theorgans of the plants of Gossypium sp. in particular the seeds reduced topowder and the seed cakes derived from them. In the case of EuphorbiaHirta it is the whole plant which is reduced to powder. The powder,which may or may not be defatted beforehand, is extracted without waterto give an aqueous solution. The aqueous solution is concentrated andtreated in different ways.

The concentrated aqueous solution is either lyophilized or convertedinto a dry powder by physical methods of evaporation and drying.

The powder thus obtained is then treated either with ethanol or withmethanol or with other organic solvents which do not dissolve thepolysaccharides.

The aqueous solution of total extracts may also be treated with ethanolor other solvents in order to precipitate the total polysaccharides.

Termination of the operations already mentioned yields all of thegalactagogue polysaccharide fractions. If necessary, a more elaboratepurification can be performed by known physical, chemical orphysicochemical methods of fractionation: ultrafiltration, filtrationthrough gel, differential precipitation, affinity chromatography, forexample.

The present invention relates more particularly to the totalpolysaccharide extracts as well as to highly active polysaccharidefractions extracted from the genus Gossypium sp. known as gossypine Aand gossypine B.

The gossypines A and B are characterized in the following manner:

The gossypines A and B are polysaccharides of molecular weight in therange 2×10⁴ -10⁵ daltons.

Gossypine A corresponds to the molecular mass fraction lying between2×10⁴ and 5×10⁴ daltons and contains 3.40%±0.3 of N and 1.32%±0.3 of S.

On acid hydrolysis gossypine A gives rise to monosaccharides which areconverted into alditols by reduction with sodium borohydride. Thesereduced monosaccharides are acetylated and analyzed by the method of gaschromatography (GC) coupled to mass spectrometry (MS). Gossypine A givesrise to the results (in analyses by GC-MS) expressed as percentages ofmonosaccharides:

    ______________________________________                                                                                 acetylated                           Rha(*) Ara     Xyl     Man   Gal   Glu   derivative                           ______________________________________                                        1.1%   5.7%    1.5%    7.6%  40.6% 41.8% 1.7%                                 ______________________________________                                         (*) by convention the symbols Rha, Ara, Xyl, Man, Gal, Glu represent the      reduced and acetylated derivatives of the corresponding sugars: rhamnose,     arabinose, xylose, mannose, galactose, glucose.                          

Gas chromatography is carried out under the following conditions:

Glass capillary column 25 m long, 0.32 m/m in diameter, film thickness:0.2 um; phase: Silar 10 C, column temperature: 220° C., injectortemperature; 220° C., interface temperature: 250° C., helium pressure:0.7 bar.

Mass spectrometry performed at 70 eV-0.25 uA, photomultiplier 1 300 eV.

Gossypine B corresponds to the molecular mass fraction lying between5×10⁴ and 10⁵ daltons and contains 6.7%±0.3 of N and 1.32%±0.3 of S.

On acid hydrolysis gossypine B gives rise to monosaccharides which areanalyzed in the form of alditol derivatives under the same conditions asin the case of gossypine A.

The following percentages of monosaccharides are found in gossypine B byanalysis on GC-MS:

    ______________________________________                                                                                 acetylated                           Rha   Ara     Xyl     Man   Gal    Glu   derivative                           ______________________________________                                        3.1%  16.3%   6.1%    2.5%  33.9%  33.3% 4.8%                                 ______________________________________                                    

The present invention also relates to extracts and to galactagogueactive fractions extracted from the genus Euphorbia Hirta Linee, namedEUPHORBINE A and EUPHORBINE B.

The euphorbines A and B are characterized in the following manner:

The euphorbines A and B are polysaccharides of molecular mass lyingbetween 3×10⁴ and 10⁵ daltons.

Euphorbine A corresponds to the molecular mass fraction lying between3×10⁴ and 5×10⁴ daltons and contains 3.10%±0.3 of N and 1.09%±0.3 of S.

On acid hydrolysis euphorbine A gives rise to monosaccharides which areconverted to the reduced form of the sugars. These reducedmonosaccharides are acetylated and analyzed by the method of gaschromatography coupled to mass spectrometry (GC-MS).

On GC-MS under the same conditions as in the case of the gossypines Aand B, euphorbine A gives the following results:

    ______________________________________                                                                                 acetylated                           Rha   Ara     Xyl     Man   Gal    Glu   derivative                           ______________________________________                                        8.4   19.5%   4.2%    3.8%  19.3%  42.6% 2.2%                                 ______________________________________                                    

Euphorbine B is a galactagogue polysaccharide fraction of molecular masslying between 5×10⁴ and 10⁵ daltons and contains 2.5%±0.3 of N and1.1±0.3 of S.

On acid hydrolysis euphorbine B give rise to monosaccharides which areconverted into the reduced form of the sugars or alditols. The alditolsobtained are acetylated and analyzed by the (GC-MS) procedure.

Euphorbine B gives the following results:

    ______________________________________                                                                                 acetylated                           Rha   Ara     Xyl     Man   Gal    Glu   derivative                           ______________________________________                                        12.7% 25.1%   9.5%    9.2%  15.8%  19.2% 8.4%                                 ______________________________________                                    

The products according to the present invention are of particularinterest for increasing the level of plasma prolactin in humans andanimals. In mammals, they increase the level of beta-casein in themammary gland.

It is possible to take advantage of these galactagogue properties in thenursing of infants and the rearing of animals, both in the form of amedicine and in the form of a food additive.

The examples given below demonstrate other properties and advantages ofthe present invention.

EXAMPLE 1

To 1000 g of powdered whole plants of Euphorbia Hirta are added 6 litersof water. The mixture is heated at 90°-95° C. for 20 minutes. It isfiltered and the solid residue is treated two more times in the samemanner. The aqueous filtrates are combined and concentrated to 2 liters.This aqueous extract is lyophilized. The lyophilizate obtained istreated with refluxing ethanol for 5 minutes (3 times 500 ml). Theinsoluble portion weighs 98.1 g and constitutes the total polysaccharideextract.

EXAMPLE 2

To 1000 g of powdered whole plants of Euphorbia Hirta are added 6 litersof water and the mixture is heated at 90°-95° C. for 20 minutes. It isfiltered and the solid residue is treated two more times in the samemanner. The aqueous filtrates are combined and concentrated to 0.3liter. 2 liters of ethanol are added and the precipitate is separated.The dried precipitate weighs 101.5 g and constitutes the totalpolysaccharide extract.

EXAMPLE 3

100 g of the total polysaccharide extract of Euphorbia Hirta aredissolved in 2 liters of water. The solution is filtered and thefiltrate is then filtered through a selectively permeable membrane witha nominal cut-off threshold at a molecular weight of 3×10⁴ daltons. Theaqueous phase containing the products of molecular mass lower than 3×10⁴daltons is lyophilized to give 21.4 g. The aqueous phase containingmolecules of molecular mass higher than 3×10⁴ daltons is ultrafilteredthrough a membrane with a cut-off threshold of 10⁵ daltons. The aqueousphase containing molecules of molecular mass lying between 3×10⁴ daltonsand 10⁵ daltons is then ultrafiltered through a membrane with a cut-offthreshold of 5×10⁴ daltons. After lyophilization or nebulization ofthese fractions 8.3 g of euphorbine A and 2.2 g of euphorbine B areobtained.

EXAMPLE 4

To 10 kg of seed powder of Gossypium sp. are added 100 liters of water.The mixture is heated at 90°-95° C. for 20 minutes. It is filtered. Thesolid residue is treated two more times with 70 liters of water eachtime at 90°-95° C. for 20 minutes. The combined filtrates yield 2.12 kgof nebulized powder of crude polysaccharides.

EXAMPLE 5

To 100 g of nebulized powder of crude polysaccharides of Gossypium sp.are added 500 ml of ethanol and the mixture is refluxed for 5 minutes.The residue is treated two more times with ethanol (2×250 ml). Theresidue insoluble in ethanol (83.3 g) constitutes the totalpolysaccharide extract.

EXAMPLE 6

To 10 kg of seed cake of Gossypium sp. are added 100 liters of water.The mixture is heated at 90°-95° C. for 20 minutes. It is filtered andthe residue is treated two more times with 70 liters of water each timeat 90°-95° C. for 20 minutes. The combined filtrates yield 1.91 g ofnebulized powder of crude polysaccharides.

EXAMPLE 7

To 100 g of nebulized powder of crude polysaccharides of Gossypium sp.of example 6 are added 500 ml of ethanol and the mixture is refluxed for5 minutes. The solid residue is treated two more times with ethanol(2×250 ml). The residue insoluble in ethanol constitutes the totalpolysaccharide extract (90.1 g).

EXAMPLE 8

20 g of the total polysaccharide extract of Gossypium sp. of example 7are dissolved in 5 liters of water. The aqueous solution isultrafiltered through a membrane with a cut-off threshold of 10⁵daltons. The fraction of molecular mass lower than 10⁵ daltons is thenultrafiltered through a membrane with a cut-off threshold of 5×10⁴daltons.

The fraction of molecular mass lower than 5×10⁴ daltons is ultrafilteredthrough a membrane with a cut-off threshold of 2×10⁴ daltons. Thesedifferent aqueous fractions are lyophilized and thus yield 295 mg ofgossypine A of molecular mass lying between 2×10⁴ and 5×10⁴ daltons and208 mg of gossypine B of molecular mass lying between 5×10⁴ and 10⁵daltons.

EXAMPLE 9 Pharmacological Studies Galactagogue Activity Principle

The administration of a galactagogue to animals is expressed by:

1) an increase in the level of beta-casein in the mammary gland;

2) an increase in the level of plasma prolactin.

Radio-Immunological Assay of Beta-Casein

Three-months old female rats are distributed in groups of four animals.The product to be studied, either in suspension or in solution in 1 mlof water, is administered by gavage twice a day for 4 days. On the 5thday the mammary glands of the sacrificed animals are removed for theradio-immunological assay of beta-casein (according to the method of F.C. GREENWOOD, W. M. HUNTER AND J. G. GLOVER, Bioch. J. (1963) 89, 114).Water is administered to control female rats. The ratio of the amount ofbeta-casein produced in the mammary glands of treated rats to thatproduced in the glands of control rats is evaluated.

The results obtained are as follows:

    ______________________________________                                                                Beta-casein in treated rats                           Dose/day Products       Beta-casein in control rats                           ______________________________________                                                 water (control)                                                                              1                                                     400 mg   product of example 4                                                                         3                                                     400 mg   product of example 5                                                                         4                                                     400 mg   product of example 6                                                                         3                                                     400 mg   product of example 7                                                                         4                                                     200 mg   product of example 1                                                                         2                                                      10 mg   gossypine A    4                                                      10 mg   gossypine B    5                                                      10 mg   euphorbine A   2                                                      10 mg   euphorbine B   4                                                     ______________________________________                                    

Radio-Immunological Assay of Prolactin

The level of plasma prolactin is assayed in ewes which have received theproducts under study by intravenous injection, using physiological serumas excipient. Blood samples are taken every 10 minutes over a period of1 hour. The plasma obtained by centrifugation is used for theradio-immunological assay of prolactin which is expressed in ng per mlof serum. The ewes serve as their own controls. The level of plasmaprolactin is assayed before and after the injection of the productsunder study. The results, expressed as the ratio between the maximumlevel of prolactin measured after injection and the base level beforeinjection, are as follows:

    ______________________________________                                        Dose/day   Products          Prolactin                                        ______________________________________                                                   physiological serum (control)                                                                   1                                                500 mg     product of example 4                                                                            4                                                300 mg     product of example 1                                                                            4                                                100 mg     gossypine A       2                                                100 mg     gossypine B       5                                                200 mg     euphorbine A      4                                                 50 mg     euphorbine B      5                                                ______________________________________                                    

I claim:
 1. A polysaccharide derived from a species of Gossypium containing monomeric units of rhamnose, arabinose, xylose, mannose, galatose, and glucose, having a molecular mass between 10,000 and 1,000,000 daltons; having 1 to 8% nitrogen and 0.8 to 2% sulfur; being substantially insoluble in ethanol; wherein said polysaccharide is selected from the group consisting of gossypine A, which has a molecular mass between 20,000 and 50,000 daltons and contains about 3.40%±0.3 by weight of nitrogen and 1.32%±0.3 by weight of sulfur, and gossypine B, which has a molecular mass of between 54,000 and 100,000 daltons and contains about 6.7%±0.3 by weight of nitrogen and 1.32%±0.3 weight of sulfur.
 2. The polysaccharide of claim 1 in the form of a dry solid.
 3. A composition for the enhancement of the secretion of prolactin in human or animal female mammals comprising an amount of the polysaccharide of claim 1 effective for the enhancement of said prolactin secretion combined with at least one diluent selected from the group consisting of pharmaceutically acceptable excipients and comestible materials. 